MECHANOCHEMISTRY OF PROTEIN 4.1S SPECTRIN-ACTIN-BINDING DOMAIN - TERNARY COMPLEX INTERACTIONS, MEMBRANE-BINDING, NETWORK INTEGRATION, STRUCTURAL STRENGTHENING

Mechanical strength of the red cell membrane is dependent on ternary i nteractions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a const itutive exon encoding 59 aa. A series of truncated SAB peptides were e ngineered to define the sequences involved in spectrin-actin interacti ons, and also membrane strength. Analysis of in vitro supramolecular a ssemblies showed that gelation activity of SAB peptides correlates wit h their ability to recruit a critical amount of spectrin into the comp lex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the ela stic skeletal network of spectrin, actin, and protein 4.1. In situ mem brane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21 -aa cassette and a conserved region near the middle of the 59 aa. Howe ver, it is shown that only weak SAB affinity is necessary for physiolo gically relevant action. Alternatively spliced exons can thus translat e into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.